We examined the role of the plasma glucose concentration per se in the secretion of counterregulatory hormones during exercise. Ten men (average age, 24 yr; maximal aerobic capacity, 31.8 mL/kg.min) were studied during two 50-min bicycle exercise periods at either normal glucose [87 +/- 1 (+/- SE) mg/dL (4.8 +/- 0.1 mmol/L)] or low glucose [59 +/- 1 mg/dL (3.3 +/- 0.1 mmol/L)]. The plasma glucose targets were achieved by exogenous insulin and variable glucose infusions. These results were compared to studies in which saline was infused. Exercise at normal glucose was associated with significant increments in plasma epinephrine (maximum 3- to 5-fold above baseline) and norepinephrine (2-fold), comparable to those that occurred during saline administration. Plasma GH increased only at the most intense exercise level, while plasma cortisol and glucagon did not increase significantly. In low glucose-exercise studies, the increase in plasma epinephrine during exercise was significantly greater than that at normal glucose (P less than 0.01), although proportional to basal preexercise levels (r = 0.73; P less than 0.001). Plasma glucagon increased almost 100%, and plasma cortisol and GH increased by 150% and 400%, respectively. Compared to the effect of the same degree of hypoglycemia in the absence of exercise, only plasma epinephrine (P = 0.002) and norepinephrine (P less than 0.001) displayed effects independent of hypoglycemia during exercise. When low glucose was reversed to normal at the midpoint of exercise, plasma epinephrine and glucagon returned to the levels obtained for the same duration of exercise at normal glucose, while norepinephrine, GH, and cortisol were only partially responsive to the rise in plasma glucose. These data suggest that 1) moderate exercise is a stimulus for a sympathoadrenal and GH response, but not a peripheral glucagon response; 2) during exercise and hypoglycemia, plasma epinephrine and norepinephrine are enhanced, while the glucagon response is entirely glucose dependent; and 3) the epinephrine response to hypoglycemia can be dissociated from that to exercise, suggesting differing control mechanisms. We conclude that the activation of counterregulatory hormones during exercise is regulated by glucose-independent mechanisms, although these responses may be augmented by concurrent hypoglycemia.